Genetic manipulation of hedgehog signaling in the endochondral skeleton reveals a direct role in the regulation of chondrocyte proliferation

Development ◽  
2001 ◽  
Vol 128 (24) ◽  
pp. 5099-5108 ◽  
Author(s):  
Fanxin Long ◽  
Xiaoyan M. Zhang ◽  
Seth Karp ◽  
Yingzi Yang ◽  
Andrew P. McMahon
Keyword(s):  
Cyclin D1 ◽  
Hedgehog Signaling ◽  
Genetic Manipulation ◽  
Bone Development ◽  
Transmembrane Protein ◽  
Wild Type ◽  
Chondrocyte Proliferation ◽  
Direct Role ◽  
Endochondral Bone ◽  
Proliferation And Differentiation

Indian hedgehog (Ihh), one of the three mammalian hedgehog (Hh) proteins, coordinates proliferation and differentiation of chondrocytes during endochondral bone development. Smoothened (Smo) is a transmembrane protein that transduces all Hh signals. In order to discern the direct versus indirect roles of Ihh in cartilage development, we have used the Cre-loxP approach to remove Smo activity specifically in chondrocytes. Animals generated by this means develop shorter long bones when compared to wild-type littermates. In contrast to Ihh mutants (Ihhn/Ihhn), chondrocyte differentiation proceeds normally. However, like Ihhn/Ihhn mice, proliferation of chondrocytes is reduced by about 50%, supporting a direct role for Ihh in the regulation of chondrocyte proliferation. Moreover, by overexpressing either Ihh or a constitutively active Smo allele (Smo*) specifically in the cartilage using the bigenic UAS-Gal4 system, we demonstrate that activation of the Ihh signaling pathway is sufficient to promote chondrocyte proliferation. Finally, expression of cyclin D1 is markedly downregulated when either Ihh or Smo activity is removed from chondrocytes, indicating that Ihh regulates chondrocyte proliferation at least in part by modulating the transcription of cyclin D1. Taken together, the present study establishes Ihh as a key mitogen in the endochondral skeleton.

EXT1 regulates chondrocyte proliferation and differentiation during endochondral bone development

Bone ◽  
2005 ◽  
Vol 36 (3) ◽  
pp. 379-386 ◽  
Author(s):  
Matthew J. Hilton ◽  
Laura Gutiérrez ◽  
Daniel A. Martinez ◽  
Dan E. Wells
Keyword(s):  
Bone Development ◽  
Chondrocyte Proliferation ◽  
Endochondral Bone ◽  
Proliferation And Differentiation

scholarly journals TGFβ and PTHrP Control Chondrocyte Proliferation by Activating Cyclin D1 Expression

2001 ◽  
Vol 12 (12) ◽  
pp. 3852-3863 ◽  
Author(s):  
Frank Beier ◽  
Zenobia Ali ◽  
Dereck Mok ◽  
Allison C. Taylor ◽  
Todd Leask ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Transcription Factor ◽  
Cyclin D1 ◽  
Cell Cycle Progression ◽  
Bone Development ◽  
Chondrocyte Proliferation ◽  
Endochondral Bone ◽  
Cycle Progression ◽  
Related Transcription Factor

Exact coordination of growth plate chondrocyte proliferation is necessary for normal endochondral bone development and growth. Here we show that PTHrP and TGFβ control chondrocyte cell cycle progression and proliferation by stimulating signaling pathways that activate transcription from the cyclin D1 promoter. The TGFβ pathway activates the transcription factor ATF-2, whereas PTHrP uses the related transcription factor CREB, to stimulate cyclin D1 promoter activity via the CRE promoter element. Inhibition of cyclin D1 expression with antisense oligonucleotides causes a delay in progression of chondrocytes through the G1 phase of the cell cycle, reduced E2F activity, and decreased proliferation. Growth plates from cyclin D1–deficient mice display a smaller zone of proliferating chondrocytes, confirming the requirement for cyclin D1 in chondrocyte proliferation in vivo. These data identify the cyclin D1 gene as an essential component of chondrocyte proliferation as well as a fundamental target gene of TGFβ and PTHrP during skeletal growth.

The CREB family of activators is required for endochondral bone development

Development ◽  
2001 ◽  
Vol 128 (4) ◽  
pp. 541-550
Author(s):  
F. Long ◽  
E. Schipani ◽  
H. Asahara ◽  
H. Kronenberg ◽  
M. Montminy
Keyword(s):  
Transgenic Mice ◽  
Bone Development ◽  
Dominant Negative ◽  
Endochondral Bone Formation ◽  
Wild Type ◽  
Growth Plate Chondrocytes ◽  
Chondrocyte Proliferation ◽  
Endochondral Bone ◽  
Proliferative Zone ◽  
Distinct Signaling Pathway

We have evaluated the importance of the CREB family of transcriptional activators for endochondral bone formation by expressing a potent dominant negative CREB inhibitor (A-CREB) in growth plate chondrocytes of transgenic mice. A-CREB transgenic mice exhibited short-limbed dwarfism and died minutes after birth, apparently due to respiratory failure from a diminished rib cage circumference. Consistent with the robust Ser133 phosphorylation and, hence, activation of CREB in chondrocytes within the proliferative zone of wild-type cartilage during development, chondrocytes in A-CREB mutant cartilage exhibited a profound decrease in proliferative index and a delay in hypertrophy. Correspondingly, the expression of certain signaling molecules in cartilage, most notably the Indian hedgehog (Ihh) receptor patched (Ptch), was lower in A-CREB expressing versus wild-type chondrocytes. CREB appears to promote Ptch expression in proliferating chondrocytes via an Ihh-independent pathway; phospho-CREB levels were comparable in cartilage from Ihh(−/−) and wild-type mice. These results demonstrate the presence of a distinct signaling pathway in developing bone that potentiates Ihh signaling and regulates chondrocyte proliferation, at least in part, via the CREB family of activators.

scholarly journals BMP2, but not BMP4, is crucial for chondrocyte proliferation and maturation during endochondral bone development

2011 ◽  
Vol 124 (20) ◽  
pp. 3428-3440 ◽  
Author(s):  
B. Shu ◽  
M. Zhang ◽  
R. Xie ◽  
M. Wang ◽  
H. Jin ◽  
...  
Keyword(s):  
Bone Development ◽  
Chondrocyte Proliferation ◽  
Endochondral Bone

scholarly journals Missense mutations in IHH impair Indian Hedgehog signaling in C3H10T1/2 cells: Implications for brachydactyly type A1, and new targets for Hedgehog signaling

2010 ◽  
Vol 15 (1) ◽  
Author(s):  
Shengzhen Guo ◽  
Jian Zhou ◽  
Bo Gao ◽  
Jianxin Hu ◽  
Hongsheng Wang ◽  
...  
Keyword(s):  
Hedgehog Signaling ◽  
Bmp Signaling ◽  
Indian Hedgehog ◽  
Endochondral Bone Formation ◽  
Missense Mutations ◽  
Loss Of Function ◽  
Endochondral Bone ◽  
Proliferation And Differentiation ◽  
Microarray Expression ◽  
Bmp Antagonist

AbstractHeterozygous missense mutations in IHH result in Brachydactyly type A1 (BDA1; OMIM 112500), a condition characterized by the shortening of digits due to hypoplasia/aplasia of the middle phalanx. Indian Hedgehog signaling regulates the proliferation and differentiation of chondrocytes and is essential for endochondral bone formation. Analyses of activated IHH signaling in C3H10T1/2 cells showed that three BDA1-associated mutations (p.E95K, p.D100E and p.E131K) severely impaired the induction of targets such as Ptch1 and Gli1. However, this was not a complete loss of function, suggesting that these mutations may affect the interaction with the receptor PTCH1 or its partners, with an impact on the induction potency. From comparative microarray expression analyses and quantitative real-time PCR, we identified three additional targets, Sostdc1, Penk1 and Igfbp5, which were also severely affected. Penk1 and Igfbp5 were confirmed to be regulated by GLI1, while the induction of Sostdc1 by IHH is independent of GLI1. SOSTDC1 is a BMP antagonist, and altered BMP signaling is known to affect digit formation. The role of Penk1 and Igfbp5 in skeletogenesis is not known. However, we have shown that both Penk1 and Igfbp5 are expressed in the interzone region of the developing joint of mouse digits, providing another link for a role for IHH signaling in the formation of the distal digits.

scholarly journals A-Raf and B-Raf Are Dispensable for Normal Endochondral Bone Development, and Parathyroid Hormone-Related Peptide Suppresses Extracellular Signal-Regulated Kinase Activation in Hypertrophic Chondrocytes

2007 ◽  
Vol 28 (1) ◽  
pp. 344-357 ◽  
Author(s):  
Sylvain Provot ◽  
Gregory Nachtrab ◽  
Jennifer Paruch ◽  
Adele Pin Chen ◽  
Alcino Silva ◽  
...  
Keyword(s):  
Cell Proliferation ◽  
Parathyroid Hormone ◽  
Bone Development ◽  
Hypertrophic Chondrocytes ◽  
Chondrocyte Proliferation ◽  
Endochondral Bone ◽  
Extracellular Signal Regulated Kinase ◽  
Chondrocyte Maturation ◽  
Extracellular Signal ◽  
Parathyroid Hormone Related Peptide

ABSTRACT Parathyroid hormone-related peptide (PTHrP) and the parathyroid hormone-PTHrP receptor increase chondrocyte proliferation and delay chondrocyte maturation in endochondral bone development at least partly through cyclic AMP (cAMP)-dependent signaling pathways. Because data suggest that the ability of cAMP to stimulate cell proliferation involves the mitogen-activated protein kinase kinase kinase B-Raf, we hypothesized that B-Raf might mediate the proliferative action of PTHrP in chondrocytes. Though B-Raf is expressed in proliferative chondrocytes, its conditional removal from cartilage did not affect chondrocyte proliferation and maturation or PTHrP-induced chondrocyte proliferation and PTHrP-delayed maturation. Similar results were obtained by conditionally removing B-Raf from osteoblasts. Because A-raf and B-raf are expressed similarly in cartilage, we speculated that they may fulfill redundant functions in this tissue. Surprisingly, mice with chondrocytes deficient in both A-Raf and B-Raf exhibited normal endochondral bone development. Activated extracellular signal-regulated kinase (ERK) was detected primarily in hypertrophic chondrocytes, where C-raf is expressed, and the suppression of ERK activation in these cells by PTHrP or a MEK inhibitor coincided with a delay in chondrocyte maturation. Taken together, these results demonstrate that B-Raf and A-Raf are dispensable for endochondral bone development and they indicate that the main role of ERK in cartilage is to stimulate not cell proliferation, but rather chondrocyte maturation.

scholarly journals Site-1 protease is essential for endochondral bone formation in mice

2007 ◽  
Vol 179 (4) ◽  
pp. 687-700 ◽  
Author(s):  
Debabrata Patra ◽  
Xiaoyun Xing ◽  
Sherri Davies ◽  
Jennifer Bryan ◽  
Carl Franz ◽  
...  
Keyword(s):  
Er Stress ◽  
Hedgehog Signaling ◽  
Endochondral Ossification ◽  
Bone Development ◽  
Active Form ◽  
Type Ii Collagen ◽  
Chondrocyte Apoptosis ◽  
Endochondral Bone Formation ◽  
Endochondral Bone ◽  
Er Stress Response

Site-1 protease (S1P) has an essential function in the conversion of latent, membrane-bound transcription factors to their free, active form. In mammals, abundant expression of S1P in chondrocytes suggests an involvement in chondrocyte function. To determine the requirement of S1P in cartilage and bone development, we have created cartilage-specific S1P knockout mice (S1Pcko). S1Pcko mice exhibit chondrodysplasia and a complete lack of endochondral ossification even though Runx2 expression, Indian hedgehog signaling, and osteoblastogenesis is intact. However, there is a substantial increase in chondrocyte apoptosis in the cartilage of S1Pcko mice. Extraction of type II collagen is substantially lower from S1Pcko cartilage. In S1Pcko mice, the collagen network is disorganized and collagen becomes entrapped in chondrocytes. Ultrastructural analysis reveals that the endoplasmic reticulum (ER) in S1Pcko chondrocytes is engorged and fragmented in a manner characteristic of severe ER stress. These data suggest that S1P activity is necessary for a specialized ER stress response required by chondrocytes for the genesis of normal cartilage and thus endochondral ossification.

scholarly journals N-3-Hydroxy Dodecanoyl-DL-homoserine Lactone (OH-dDHL) Triggers Apoptosis of Bone Marrow-Derived Macrophages through the ER- and Mitochondria-Mediated Pathways

2021 ◽  
Vol 22 (14) ◽  
pp. 7565
Author(s):  
Kyungho Woo ◽  
Dong Ho Kim ◽  
Man Hwan Oh ◽  
Ho Sung Park ◽  
Chul Hee Choi
Keyword(s):  
Bone Marrow ◽  
Quorum Sensing ◽  
Deletion Mutant ◽  
Transmembrane Protein ◽  
Cell Communication ◽  
Homoserine Lactone ◽  
Host Responses ◽  
Wild Type ◽  
Mutant Strains

Quorum sensing of Acinetobacter nosocomialis for cell-to-cell communication produces N-3-hydroxy dodecanoyl-DL-homoserine lactone (OH-dDHL) by an AnoR/I two-component system. However, OH-dDHL-driven apoptotic mechanisms in hosts have not been clearly defined. Here, we investigated the induction of apoptosis signaling pathways in bone marrow-derived macrophages treated with synthetic OH-dDHL. Moreover, the quorum-sensing system for virulence regulation was evaluated in vivo using wild-type and anoI-deletion mutant strains. OH-dDHL decreased the viability of macrophage and epithelial cells in dose- and time-dependent manners. OH-dDHL induced Ca2+ efflux and caspase-12 activation by ER stress transmembrane protein (IRE1 and ATF6a p50) aggregation and induced mitochondrial dysfunction through reactive oxygen species (ROS) production, which caused cytochrome c to leak. Pretreatment with a pan-caspase inhibitor reduced caspase-3, -8, and -9, which were activated by OH-dDHL. Pro-inflammatory cytokine and paraoxonase-2 (PON2) gene expression were increased by OH-dDHL. We showed that the anoI-deletion mutant strains have less intracellular invasion compared to the wild-type strain, and their virulence, such as colonization and dissemination, was decreased in vivo. Consequently, these findings revealed that OH-dDHL, as a virulence factor, contributes to bacterial infection and survival as well as the modification of host responses in the early stages of infection.

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